Isolation of Metarhizium anisopliae carboxypeptidase A with native disulfide bonds from the cytosol of Escherichia coli BL21(DE3)

• Published in: Protein expression and purification 2012-03, Vol.82 (1), p.116-124
• Main Authors: Austin, Brian P., Waugh, David S.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Baculoviridae - genetics; Carboxypeptidase; Carboxypeptidases A - chemistry; Carboxypeptidases A - genetics; Carboxypeptidases A - isolation & purification; Carboxypeptidases A - metabolism; Disulfides - chemistry; Disulfides - metabolism; DsbA; DsbC; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - isolation & purification; Escherichia coli Proteins - metabolism; Gene Expression; Genetic Vectors - genetics; gor; His-tag; Maltose-binding protein; Maltose-Binding Proteins - chemistry; Maltose-Binding Proteins - genetics; Maltose-Binding Proteins - isolation & purification; Maltose-Binding Proteins - metabolism; MeCPA; Metarhizium - chemistry; Metarhizium - enzymology; Metarhizium - genetics; Metarhizium - metabolism; Metarhizium anisopliae; Molecular Sequence Data; Polyhistidine tag; Protein disulfide isomerase; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Solubility; Thermolysin; trxB
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2011.11.015

► We describe the production of a fungal carboxypeptidase in the cytosol of Escherichia coli. ► The zymogen is kept soluble by fusing it to E. coli maltose-binding protein. ► Co-expression of DsbC in the cytosol promotes the formation of disulfide bonds. ► The zymogen is activated by digestion with thermolysin. ► Active carboxypeptidase is purified by affinity and conventional chromatography. The carboxypeptidase A enzyme from Metarhizium anisopliae (MeCPA) has broader specificity than the mammalian A-type carboxypeptidases, making it a more useful reagent for the removal of short affinity tags and disordered residues from the C-termini of recombinant proteins. When secreted from baculovirus-infected insect cells, the yield of pure MeCPA was 0.25mg per liter of conditioned medium. Here, we describe a procedure for the production of MeCPA in the cytosol of Escherichia coli that yields approximately 0.5mg of pure enzyme per liter of cell culture. The bacterial system is much easier to scale up and far less expensive than the insect cell system. The expression strategy entails maintaining the proMeCPA zymogen in a soluble state by fusing it to the C-terminus of maltose-binding protein (MBP) while simultaneously overproducing the protein disulfide isomerase DsbC in the cytosol from a separate plasmid. Unexpectedly, we found that the yield of active and properly oxidized MeCPA was highest when coexpressed with DsbC in BL21(DE3) cells that do not also contain mutations in the trxB and gor genes. Moreover, the formation of active MeCPA was only partially dependent on the disulfide-isomerase activity of DsbC. Intriguingly, we observed that most of the active MeCPA was generated after cell lysis and amylose affinity purification of the MBP-proMeCPA fusion protein, during the time that the partially purified protein was held overnight at 4°C prior to activation with thermolysin. Following removal of the MBP-propeptide by thermolysin digestion, active MeCPA (with a C-terminal polyhistidine tag) was purified to homogeneity by immobilized metal affinity chromatography (IMAC), ion exchange chromatography and gel filtration.